The biosynthesis of two flavoproteins, 6-hydroxy-D-nicotine oxidase with covalently bound FAD and 6-hydroxy-L-nicotine oxidase containing non-covalently bound FAD, was studied in wild-type cells and in a riboflavin-requiring mutant of Arthrobacter oxidans. In the mutant cells, the rate of synthesis and the maximal activity level of both enzymes after induction by nicotine depended on the amount of added riboflavin. The low rate of synthesis in the presence of 2 micron riboflavin could be enhanced during the induction phase by further addition of riboflavin (33 micron). Inhibitors of translation (chloramphenicol or streptomycin) completely blocked the synthesis of both flavoproteins. Inhibitors of transcription (rifamycin S or actinomycin D) stopped the synthesis of both enantiozymes in wild-type cells and in the mutant grown in the presence of a saturating supply of riboflavin (15 micron). Under conditions of restricted flavoprotein synthesis (2 micron riboflavin in the medium), however, the mutant cells continued to synthesize the enzyme for 2--3 h after the addition of the transcription inhibitors. It appears, that in these cells a rather stable m-RNA accumulated during riboflavin-limited flavoprotein synthesis. The dependence of the effect of transcription inhibitors on the extracellular supply of riboflavin suggests that the regulation of the synthesis of both flavoproteins occurs not only by control of gene expression (induction by nicotine), but also at the level of translation through the availability of FAD.

6‐Hydroxy‐d‐nicotine oxidase is an inducible flavoprotein of Arthrobacter oxiduns in which one mole of FAD is bound covalently to the polypeptide chain. During cell‐free translation of poly‐ somes from nicotine‐induced A. oxidans cells in the presence of an Escherichia coli (MRE 600) supernatant fraction, labelled FAD, leucine and histidine were incorporated into 6‐hydroxy‐d‐nicotine oxidase in the same ratio found in the enzyme isolated from whole cells. This indicates that one mole FAD is covalently attached per mol of 6‐hydroxy‐d‐nicotine oxidase synthesized in vitro. In the native enzyme the coenzyme molecule is bound via its 8α‐methyl group to the N3 atom of a histidyl residue. From the translation products an aminoacyl‐riboflavin was isolated and its identity with synthetic 8α‐(N3‐histidyl)‐riboflavin was shown.

The process, by which FAD is attached covalently to the 6-hydroxy-~-nicotine oxidase apoprotein in D-nicotine-induced cells of Arthrobucter oxiduns was studied in vitro.[3H]Adenine-labelled FAD prepared biosynthetically in Clostridium kluyveri was incorporated into the 6-hydroxy-~-nicotine oxidase molecule during cell-free translation. FAD rather than FMN or riboflavin was thus shown to be the flavin derivative transferred to the polypeptide chain. After short-term protein synthesis on ribosomes from induced A . oxiduns cells in the presence of an Escherichiu coli MRE 600 supernatant fraction and [~denine-2-~H]FAD, the peptidyl-tRNA fraction was separated from completed polypeptides. Labelled FAD was found to be covalently attached to the tRNA-bound polypeptides. Cleavage of the tRNA-peptide bond released labelled polypeptides the largest of which migrated as authentic 6-hydroxy-~-nicotine oxidase during dodecylsulfate/polyacrylamide gel electrophoresis. These results strongly suggest that FAD is incorporated into the nascent polypeptide chains of 6-hydroxy-~-nicotine oxidase during ribosomal translation.In the last few years an increasing number of flavoproteins have been reported to contain covalently bound coenzymes (for review see [l]). However, the flavin-binding process during the biosynthesis of these proteins is not yet understood. In Arthrobucter oxidans, 6-hydroxy-~-nicotine oxidase is induced in the presence of D-nicotine [2]; the coenzyme, FAD, was shown [3] to be covalently attached through its 8cc-methyl group to N-3 of a histidyl residue of the apoprotein. This enzyme is synthesized during the early stationary phase of growth [4] where only a limited number of proteins is assumed to be produced. Studies with a riboflavin-requiring mutant on the flavin-binding process in vivo indicated that the function of intact ribosomes is necessary for the formation of enzymatically active holoenzyme, even during flavin-limited 6-hydroxy-~-nicotine oxidase synthesis With isolated polysomes and the translation system of the ribonuclease-free strain (MRE 600) of Esche-151.

The mechanism of in vitro inactivation and ATP-dependent rapid reactivation of rat liver tyrosine aminotransferase by a membrane-bound system from rat liver and kidney cortex and the nucleotide specificity of this process was investigated using partially purified tyrosine amino­ transferase as a substrate. Adenosine 5′-triphosphate (ATP) could be replaced by guanosine 5′-tri-phosphate (GTP), whereas inosine 5′-triphosphate (ITP) was less effective. During reactivation [γ-32P]A T P was incorporated into the enzyme and not excorporated by incubation of the labeled enzyme with excess non-radioative ATP. Inactivation of labeled tyrosine aminotransferase by a particulate fraction led to a decrease protein-bound radioactivity concomitant with an increase of [32P] orthophosphate. This points to a phosphorylation and dephosphorylation mechanism in the regulation of tyrosine aminotransferase activity.

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